Sandbox 34

= Lysozyme =

Introduction
Lysozyme is an amazingly effective enzyme known for its catalytic activity of lysing bacterial cell walls. Lysozyme is effective at this through its ability to hydrolyze the 1,4 beta-linkages from the N-acetylmuramic acid to the N-acetylglucosamine residues in bacterial cell walls[3]. Lysozyme also contains a unique ability to hydrolyze the bonds in chitin, which can be found in things like tough outer exoskeletons in crustaceans and in fungi. Although its found in several different types its most predominate and widely used type is from egg whites. A Space Filled Model of the 1hew hen egg white lysozyme comprised of all its elements and its small compact 129 residues can be found in the applet below.

We are most familiar with lysozyme's powerful antibacterial character as is seeks to attack peptidoglycans within the cell walls of most Gram-positive bacteria. It is because of this ability that we find lysozyme as a part of the innate immune in the human body as one of the first defenses towards warding off infections. We can find them also externally in such secretions for the same reasons such as saliva and tears[4].



= Lysozyme's Secondary Structures =

Lysozyme is a small but complex structure that utilizes all of its resources in its Secondary Structures. Lysozyme contains both five alpha helices as well as five beta sheets[2]. As the structures can be seen, the alpha helices (in the pink), are grouped together fairly tightly while the majority of three out of the five beta sheets (the yellow arrows), group with themselves. The two other beta sheets are paired off together separately on the other side as you can observe. The beta sheets as seen bind very tightly as they line up directly next to each other as a result of their anitparallel motif. This allows them to maximize their hydrogen bonding while still staying free of torsional and steric strains[1]. The secondary structures can also be viewed in the Space Filled Model the right to help get a greater picture of how they fit together in the proteins overall shape as a whole.



= Protein Folding =

Lysozyme’s unique tertiary structure resides from the strong forces that regulate is folding. It is because lysozyme contains both hydrophobic (water hating) and hydrophilic (water loving) regions that it is controlled by the hydrophobic effects and is driven by hydrophobic collapse. This major driving force referred to as hydrophobicity requires the hydrophobic regions that cannot interact with water to be driven into the internal core of the protein so that it is shielded from all water molecules[1]. This leaves the entire outside of the protein to be comprised of its hydrophilic regions, which are free to interact with all the water molecules the protein encounters in solution. The model to your left demonstrates this as it has been rendered transparent in a space fill model demonstrating the Reaction with Water. Notice how the red hydrogens from neighboring water molecules may adhere to the outside however, they are kept far away from the proteins hydrophobic core on the inside[1].

Hydrophobic Effect
As seen to the left, the Hydrophobic Regions can be observed by looking at the differences between the two colors on the protein structure. You can notice that the gray colors represent the hydrophobic regions as they remain tucked away on the inside of the protein. Incasing them would be the hydrophilic regions that surround the protein and are represented by the purple color.

= Polar and Nonpolar Residues =

Another major driving factor behind lysozyme’s folding pathway is its polar and nonpolar residues. These Charged Residues can be observed above as the charges they carry with them are positioned on the outside of the protein for stability and the ability to react with water[2]. In this applet, the blue portions can be assumed as the positive or cationic residues while the red areas can be assumed as the negative charged residues or the anionic areas. The nonpolar residues are unable to be examined in this view as they are thrust into the center of the internal core of the protein where they are safe from reacting with the pink and purple polar areas and water molecules on the outside. A model that shows Polarity and Charges through the model can be found above as well. Here we can look through the model and understand how the polar and charged residues are found only on the outside of the protein where they are left to react.



= Structural components and ligands =

Lysozyme’s enzymatic ability is driven from its unique active sites and its intermolecular structure that holds it together. Lysozyme, although a small protein, is very stable as it is held together by four disulfide bonds. The disulfide bonds are derived from the double bonds formed between two cysteine groups and help regulate the proteins structural integrity as it is folded into its tertiary structure[4][1]. These Disulfide Bonds can be viewed to your right as the yellow bonds set across the backbone and side chains within the structure. Lysozyme is also structurally held together by a very intricate series of Hydrogen bonds within which contribute to its intermolecular forces and integrity as well as its ability to react with other molecules on the outside of its side chains. The Hydrogen Bonds can also be viewed as the white bonds connecting the structural elements as it is folded.

Ligand
Lysozyme’s Ligand as seen to your right, is also another extremely important part for its function. It allows for it to bind to the substrate the way it specifically requires in order to proceed enzymatically. The diagram of the ligand to your right depicts the protein in the brownish color surrounded by the blue substrate and the ligand in light green.

Active Site
It has been shown through X-Ray Crystallography that the Glu 35 and Asp 52 residues are lysozyme’s predominate catalytic side chains[1]. In the hydrolysis process the Asp 52 residue acts as a nucleophile while the Glu 35 participates in acid base reactions[1]. It is because of these residues that lysozyme is able to effectively bind to the desired substrate. The Active Site close up can be found to your left using a ball and stick view on the side chains.

= Binding and Catalytic Function =

As mentioned above, Lysozyme is found to hold remarkable anti-bacterial properties which allow for it to lyse the cell walls of most Gram-positive bacteria. Catalytically lysozyme is a very tailored and specific enzyme that requires just the right six ringed hexasaccharide sugar unit to bind to[1]. Lysozyme’s hydrolysis of the glycosidic 1,4 beta-linkages from the N-acetylmuramic acid to the N-acetylglucosamine residues first start with its ligand binding to the sugar and distorting the D residue of the sugar from its torsional strain. The D ring is sterically strained and is then forced into a half chair conformation where it stresses the bonds between itself and the E ring. The Glu 35 residue from the lysozyme participates in acid base catalysis and helps donate a proton to stabilize it. From here the Asp 52 group of the lysozyme acts as a nucleophile and attacks the carbon on the D group producing an intermediate site as water soon replaces it and binds with the E site breaking the glycosidic bond on the polysaccharide chain[1].

= References=

[1] Voet, Donald, Voet, G Judith, Fundamentals of Biochemisty: Life at the molecular level

[2] Lysozyme structure. 3D pictures of Lysozyme structure: http://lysozyme.co.uk/lysozyme-structure.php

[3] Lysozyme: http://lysozyme.co.uk/

[4] Lysozyme,24 December 2001 http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/L/Lysozyme.html

[5] Harald Schwalbe1, Shaun B. Grimshaw1, Andrew Spencer2, Matthias Buck1 A refined solution structure of hen lysozyme determined using residual dipolar coupling http://onlinelibrary.wiley.com/doi/10.1110/ps.43301/full